556 T. HoRio, J. Yamashita, T. Vamanaka, M. Nozaki and K. Okunuki 



the substrates are oxidized by Y-LDH: lactate, very strong; a-hydroxy- 

 butyrate, a little weaker than lactate; malate, very weak. Throughout the 

 time course of the denaturation of Y-LDH by heat or by lowering the pH 

 in the presence of either lactate of a-hydroxybutyrate, the reactivity of the 

 enzyme toward the two substrates maintains a constant ratio. This is the 

 case even if the activity is assayed with the use of phenazine methosulphate, 

 ferricyanide, methylene blue, or cytochrome c (Yamashita, unpublished). 

 This fact indicates that lactates and a-hydroxybutyrate are dehydrogenated 

 not only by the same enzyme (Y-LDH), but also by the same dehydrogenase 

 moiety (reaction (1)). Since Y-LDH shows much lower affinities for the 

 other substrates as compared with lactate and a-hydroxybutyrate, it has been 

 found difficult to apply tliis method to the dehydrogenation of the other 

 substrates. 



DISCUSSION 

 Boeri, Cutolo, Luzzati and Tosi (1955) and Boeri and Tosi (1956) have 

 found that their best preparation of Y-LDH contains one FMN residue, one 

 haem group and eight iron atoms not bound to haem for each 230,000 g 

 protein, and they demonstrated the absence of a diphosphopyridine 

 nucleotide-dependence for the dehydrogenase activity. Therefore they 

 proposed the following scheme for electron transport in Y-LDH : 



Lactate —> FMN — >■ non-haem irons > haem iron — ^ cytochrome c 



(Y-LDH) 



The extensive characterization of the crystalline Y-LDH by Appleby and 

 Morton (see Morton, 1955, 1958; Appleby and Morton, 1954, 1959a, b) may 

 require some modifications of the scheme as follows : 



Lactate —> riboflavin phosphate > haem iron — '-> cytochrome c 



(Y-LDH) 



Moreover, the successful crystallization of Y-LDH may support the concept 

 that both dehydrogenation of an organic compound and the transport of 

 the resulting electrons to a cytochrome can be carried out by one enzyme. 



The linkage between the dehydrogenation of succinate and the reduc- 

 tion of cytochrome h may fit this concept. Singer and co-workers (Singer, 

 Kearney and Bernath, 1956; Singer, Massey and Kearney, 1957) have 

 succeeded in preparing mammalian succinate dehydrogenase in a truly homo- 

 geneous, water-soluble state. Their dehydrogenase contains flavin dinucleo- 

 tide and non-haem iron(s). The purest enzyme can oxidize succinate with 

 the aid of phenazine methosulphate, but not with methylene blue and 



